Histone is a basic protein ion-bonded to genomic DNA, which is commonly present in the nucleus of eukaryotic cells of from multicellular organisms including human to unicellular organisms represented by fungus (mold, yeast). Histone generally consists of 5 kinds of components (H1, H2A, H2B, H3 and H4), which are highly similar beyond biological species. In the case of histone H4, for example, budding yeast histone H4 (full-length 102 amino acid sequence) and human histone H4 (full-length 102 amino acid sequence) are identical in 92% of the amino acid sequences and differ only in 8 residues. Among the natural proteins assumed to be present in several tens of thousand kinds in one organism, histone is known to be a protein most highly preserved among eucaryotic species. Genomic DNA is folded due to a regular bond to the histone, and a complex of the both forms a basic structural unit called nucleosome. Then, coagulation of the nucleosomes forms a chromosomal chromatin structure. Histone is subject to modification such as acetylation, methylation, phosphorylation, ubiquitination, sumolation and the like at an N-terminal portion called a histone tail, and maintains or specifically converts the chromatin structure to control reactions occurring on chromosomal DNA such as gene expression, DNA replication, DNA repair and the like. Post-translational modification of histone is an epigenetic regulatory mechanism, and is considered essential for the gene regulation of eukaryotic cells. For example, acetylation of histone is controlled by a pair of modification enzymes (i.e., histone acetylation enzyme and deacetylation enzyme). Generally, deacetylation enzymes act dominantly, and histone is maintained in a deacetylated state. When a cell is activated by stimulation, histone acetylation enzyme acetylates amino group of the lysine residue of histone and neutralizes the positive charge of the amino group. As a result, the interactions between nucleosomes become loose and transcription factor is recruited to start the transcription.
As a domain structure of proteins bound to acetylated lysine of histone, bromodomain is known. Humans have thirty-some kinds of bromodomain-containing proteins. Among them, BRD2, BRD3 and BRD4 are the proteins interacting with acetylated histone H3/H4. Among them, BRD4 is known to be a protein involved in the cell cycle and gene expression (non-patent document 1: Nature 399, 491-496, 1999) (non-patent document 2: JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 282 No. 18 13141-13145, 2007). BRD4 belongs to a BET (bromodomain and extraterminal) family protein having two bromodomains and one extraterminal domain in a molecule. As the BET family proteins other than BRD4, BRD2, BRD3 and BRDt derived from human are known. Heretofore, a compound that inhibits binding between such BET family proteins and acetylated histone is not known.
In connection with the acetylation of histone, a compound inhibiting histone deacetylation enzyme is known to show cell cycle discontinuation, differentiation induction and apoptosis induction activity on tumor cells (non-patent document 3: Exp. Cell Res., 177, 122-131, 1988, non-patent document 4: Cancer Res., 47, 3688-3691, 1987). However, there is no report on whether or not a compound inhibiting binding between acetylated histone and a bromodomain-containing protein influences the tumor cells.
In recent years, there are some cases where BRD4-NUT fusion protein is expressed in epithelial cell carcinoma (midline carcinoma) in the upper tissue in the body such as thymus, airway, lung and the like. Patients showing expression of such fusion protein are known to resist radiation treatment and chemical therapy, and show poor prognosis (non-patent document 6: Cancer Research vol. 63 Jan. 15, 2003 p304-307, non-patent document 7: Journal of clinical oncology Vol. 22 No. 20 Oct. 15, 2004 p4135-4139). In addition, it has been reported that, in midline carcinoma, t(9;15) chromosomal translocation of chromosome 9 and chromosome 15 also forms fusion protein BRD3-NUT of BRD3 protein and NUT protein. It has been reported that, in the cancer cell lines derived from patients expressing each of BRD3-NUT fusion protein and BRD4-NUT fusion protein, genetic inhibition of the expression of the fusion proteins by siRNA results in the discontinuation of the growth of the cancer cells (non-patent document 8: Oncogene advance online publication 15 Oct. 2007; doi: 10.1038/sj.onc.1210852). Hence, a medicament inhibiting the function of such fusion proteins is expected to be an antitumor agent. However, there is no report teaching that inhibition of binding between acetylated histone and bromodomain present on the fusion protein inhibits the functions of these fusion proteins.
On the other hand, it is known that a thienotriazolodiazepine compound represented by the following formula (I)
wherein
R1 is alkyl having a carbon number of 1-4,
R2 is a hydrogen atom; a halogen atom; or alkyl having a carbon number of 1-4 optionally substituted by a halogen atom or a hydroxyl group,
R3 is a halogen atom; phenyl optionally substituted by a halogen atom, alkyl having a carbon number of 1-4, alkoxy having a carbon number of 1-4 or cyano; —NR5—(CH2)m—R6 wherein R5 is a hydrogen atom or alkyl having a carbon number of 1-4, m is an integer of 0-4, and R6 is phenyl or pyridyl optionally substituted by a halogen atom; or —NR7—CO—(CH2)n—R8 wherein R7 is a hydrogen atom or alkyl having a carbon number of 1-4, n is an integer of 0-2, and R8 is phenyl or pyridyl optionally substituted by a halogen atom, and
R4 is —(CH2)a—CO—NH—R9 wherein a is an integer of 1-4, and R9 is alkyl having a carbon number of 1-4; hydroxyalkyl having a carbon number of 1-4; alkoxy having a carbon number of 1-4; or phenyl or pyridyl optionally substituted by alkyl having a carbon number of 1-4, alkoxy having a carbon number of 1-4, amino or a hydroxyl group or —(CH2)b—COOR10 wherein b is an integer of 1-4, and R10 is alkyl having a carbon number of 1-4,    has a cell adhesion inhibitory action and is useful for inflammatory intestine diseases, and has an action inhibiting costimulatory signals from CD28 on T cells and is useful for the rejection during transplantation, autoimmune diseases and allergic diseases (patent document 1: WO 98/11111, patent document 2: WO 2006/129623). However, it is not known at all that these compounds have an action to inhibit binding between acetylated histone and BET family protein, and an antitumor action.    patent document 1: WO 98/11111    patent document 2: WO 2006/129623    non-patent document 1: Nature 399, p491-496, 1999    non-patent document 2: JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 282 No. 18 p13141-13145, 2007    non-patent document 3: Exp. Cell Res., 177, p122-131, 1988    non-patent document 4: Cancer Res., 47, p3688-3691, 1987    non-patent document 5: American Journal of Pathology Vol. 159 No. 6, p1987-1992 December 2001    non-patent document 6: Cancer Research vol. 63, p304-307 Jan. 15, 2003    non-patent document 7: Journal of clinical oncology Vol. 22 No. 20, p4135-4139 Oct. 15, 2004    non-patent document 8: Oncogene advance online publication 15 Oct. 2007; doi: 10.1038/sj.onc.1210852